Combination therapy

ABSTRACT

Reversing resistance to a B-Raf inhibitor for the treatment of a proliferative disease by obtaining a tumor sample from the patient and testing it for genetic alterations in a panel of genes comprising BRAF, CRAF, CCND1, CDK4, HER2, IGF-1R, cMET, FGFR1, FGFR2, FGFR3 EGFR, MAP2K1, MAP2K2, NRAS, KRAS HRAS, PTEN, PIK3CA, and P16 and administering a drug combination therapy comprising the B-Raf inhibitor and a second inhibitor which overcomes resistance to the B-Raf inhibitor, which second inhibitor is selected based on genetic alterations discovered in the tumor sample.

RELATED APPLICATIONS

This application is a 371 filing of Patent Cooperation TreatyApplication No. PCT/IB2014/055975, filed Mar. 19, 2014, which claimspriority to U.S. Patent Application No. 61/804,056, filed Mar. 21, 2013,the contents of which are incorporated herein by reference in theirentirety.

SUMMARY

The present invention relates to the use of a B-Raf inhibitor incombination with a second inhibitor for the treatment of a patientsuffering from a proliferative disease characterized by a mutation inB-Raf, wherein the second inhibitor is selected based on geneticalterations identified in a tumor sample.

BACKGROUND

Important advances have been made in the understanding of the molecularchanges associated with the development of melanoma. Oncogenic mutationsof B-RAF, a serine-threonine protein kinase in the RAF/MEK/ERK pathway,are particularly common in melanoma, with 40 to 60% of melanoma carryingan activating mutation in the B-Raf gene. The substitution of glutamicacid for valine at amino acid 600 (V600E mutation) represents more than95% of the reported B-Raf mutations. This mutation constitutivelyactivates B-Raf and downstream signal transduction in the RAF/MEK/ERKpathway, which signals for cancer cell proliferation and survival. Inaddition to melanoma, such mutations of B-Raf are known to occur inother proliferative diseases, for example, colorectal cancer, thyroidcancer, particularly papillary thyroid cancer, astrocytomas, pancreaticcancer, and neurofibromatosis. Although dramatic results are known tooccur when such diseases are treated with a B-Raf inhibitor, thedevelopment of resistance to treatment with the B-Raf inhibitor istypical, often occurring within a fairly short period of time.

There are multiple paths to resistance to treatment with a B-Rafinhibitor. The main mechanisms result in reactivation of the RAF/MEK/ERKsignaling pathway in the presence of the B-Raf inhibitor. Thisreactivation can occur via increased activity of receptor tyrosinekinases (RTKs) via gene amplification, and over expression and/or ligandproduction, acquisition of mutations in the NRAS and MEK1 genes, bypassof BRAF via over-expression of kinases such as COT and RAF-1 (CRAF),expression of splice variants of the mutant BRAF allele, and increasedexpression of the mutant BRAF allele due to, e.g. gene amplification. Inaddition, activation of survival pathways such as the PIK3Cα signalingsystem that are distinct from the MAPK pathway, either via activation ofRTKs such as PDGFR-β and IGF-1R or loss of the PTEN gene may also play arole in resistance. Other mechanisms, through c-MET and the FGFR familyof RTKs, are potential mechanisms that may promote resistance to B-Rafinhibitors in multiple melanoma.

The findings described above highlight the importance of identifyingmechanisms of resistance in real time, in order to initiate a rationalcombination therapy early on after relapse on B-Raf inhibitor treatment.Using a mechanism-based approach with the comparison of the geneticalterations present in a patient's tumor at the time of relapse versuspre-treatment, it should be possible to identify likely resistancemechanisms. This will help selecting the appropriate drug combinationtherapy for an individual patient in order to better circumventresistance. The present invention relates to a mechanism-basedcombination treatment approach to expand and improve the therapeuticoptions for patients with BRAF-mutant advanced or metastatic melanomathat have very poor prognosis after the development of resistance toB-Raf inhibitors.

BRIEF DESCRIPTION

The present invention relates to treating a patient suffering from aproliferative disease characterized by a mutation in B-Raf with a B-Rafinhibitor wherein resistance to the B-Raf inhibitor is reduced by

(a) determining genetic alterations in a tumor sample taken from thepatient,

(b) administering a drug combination therapy consisting of the B-Rafinhibitor and a second inhibitor to the patient, wherein the secondinhibitor is selected based on the genetic alterations found in thetumor sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—shows the effect of the Compound of Formula (I) and Compound F assingle agents and in combination on the growth of the HT-29 cell linemodel in vivo as described in Example 4.

FIG. 2—shows the effect of the Compound of Formula (I) and Compound F assingle agents and in combination on the growth of the RKO cell linemodel in vivo as described in Example 4.

FIG. 3—shows the effect on proliferation of combining the RAF inhibitor(Compound of Formula I) with the FGFR inhibitor Compound H in twomelanoma derived cell lines that harbor the BRAFV600E-encoding allele ofBRAF. Shown is the growth in real time of the (Top A) COLO 741 and(Bottom B) SK-MEL-5 cell lines as measured using the xCELLigenceimpedance-based cell analyzer as discussed in Example 5. Where indicatedFGF2 and Compound H were supplemented to the media at concentrations of100 ng/ml, and 1 uM, respectively. The Compound of Formula (I) was usedat 500 nM in (A) and 100 nM (B).

FIG. 4—The effect on signaling of combining the Compound of Formula (I)with the FGFR inhibitor Compound H and the FGFR ligand FGF2 in twoBRAFV600E mutant melanoma-derived cell lines in vitro. Shown is westernanalysis of both phosphorylated and total AKT, ERK1/2, and MEK1/2proteins isolated from (A) COLO 74 and (B) SK-MEL-5 cells followingtreatment with the Compound of Formula (I) (100 nM), FGF2 (100 ng/ml),and Compound H (1 uM). Cells were treated for 2 and 24 hours with agentssingly and in combination as discussed in Example 5.

DETAILED DESCRIPTION

The present invention relates to a method for treating a patientsuffering from a proliferative disease characterized by a mutation inB-Raf, particularly a V600 mutation in B-Raf, which comprises:

(a) obtaining a tumor sample from the patient and testing for a geneticalteration in a panel of genes comprising BRAF, CRAF, CCND1, CDK4, HER2,IGF-1R, cMET, FGFR1, FGFR2, FGFR3 EGFR, MAP2K1, MAP2K2, NRAS, KRAS HRAS,PTEN, PIK3CA, and P16.

(b) administering a drug combination therapy comprising a B-Rafinhibitor and a second inhibitor, which second inhibitor is selectedbased on genetic alterations discovered in the tumor sample.

In one embodiment, the proliferative disease is cancer. The term“cancer” is used herein to mean a broad spectrum of tumors, includingall solid tumors and hematological malignancies. Examples of such tumorsinclude but are not limited to benign or malignant tumors of the brain,lung (in particular small-cell lung cancer and non-small cell lungcancer), squamous cell, bladder, gastric, pancreatic, breast, head andneck, renal, kidney, ureter, ovarian, prostate, colorectal, esophageal,testicular, gynecological (e.g., uterine sarcomas, carcinoma of thefallopian tubes, endometrial, cervix, vagina or vulva), thyroid,pancreatic, bone, skin, melanoma, uterine, ovarian, rectal, anal, colon,testicular, Hodgkin's disease, esophageal, small intestine, endocrinesystem (e.g., thyroid, parathyroid, or adrenal glands), sarcomas of softtissues, urethra, penis, leukemia, lymphomas, neoplasms of the centralnervous system, sarcomas, myeloma, biliary, liver, neurofibromatosis,acute myelogenous leukemia (AML), myelodysplastic syndromes (MDS), andKaposi's sarcoma.

In a further embodiment of the present invention, the proliferativedisease is melanoma, lung cancer (including non-small cell lung cancer(NSCLC)), colorectal cancer (CRC), breast cancer, kidney cancer such ase.g., renal cell carcinoma (RCC), liver cancer, endometrial cancer,acute myelogenous leukemia (AML), myelodysplastic syndromes (MDS),thyroid cancer, particularly papillary thyroid cancer, pancreaticcancer, neurofibromatosis or hepatocellular carcinoma.

In a further embodiment of the present invention, the proliferativedisease is a solid tumor. The term “solid tumor” especially meansmelanoma, breast cancer, ovarian cancer, colorectal cancer, andgenerally gastrointestinal tract, cervix cancer, lung cancer (includingsmall-cell lung cancer and non-small cell lung cancer), head and neckcancer, bladder cancer, prostate cancer or Kaposi's sarcoma.

More particularly, the present invention relates to a method fortreating a patient suffering from a proliferative disease characterizedby a V600 mutation in B-Raf, for example a V600E mutation. Proliferativediseases frequently characterized by such a mutation include melanoma,colorectal cancer, thyroid cancer, particularly papillary thyroidcancer, astrocytomas, pancreatic cancer, and neurofibromatosis. Thepresent invention especially relates to such a method wherein theproliferative disease is melanoma characterized by a V600 mutation inB-Raf, for example a V600E, V600K or V600G mutation.

B-Raf inhibitors and their use for treating proliferative diseases areknown in the art. Vemurafenib (PLX4032) is a BRAF inhibitor which wasapproved by the FDA for the treatment of patients with melanoma whosetumors express BRAF V600E. Sorafenib and dabrafenib and CEP-32496 areadditional known B-Raf inhibitors. The benzimidazolyl pyridyl ethers,disclosed in U.S. Pat. No. 7,482,367, which is here incorporate byreference in its entirety, are B-Raf inhibitors useful in the presentcombinations, particularly RAF265. The pyrrazole pyrimidines, which aredisclosed in WO 2011/025927 and which is here incorporate by referencein its entirety, are another class of B-Raf inhibitors useful for thepresent combinations.

An appropriate second inhibitor to be combined with the B-Raf inhibitoris selected in accordance with Table 1 for treatment of the patientbased on the genetic alterations found in the tumor sample. The geneticalterations can result from amplification of a gene, mutations in a geneor loss of the gene's activity.

TABLE 1 Drug to be given in combination Genetic Alterations with theB-Raf Amplification Mutation Loss inhibitor BRAF MAP2K1 Mek1/2 inhibitorCRAF MAP2K2 EGFR NRAS KRAS HRAS CCND1 CDK4 P16 CDK 4 inhibitor CDK4 HER2PTEN PTEN PI3 Kinase IGF-1R PIK3CA inhibitor cMET c-Met receptortyrosine kinase inhibitor FGFR1 FGFR kinase FGFR2 inhibitor FGFR3 Or noalteration in any of the above Mek1/2 inhibitor identified genes

The information relating to the genes identified in Table 1, theirsequences and associated proteins are known to those of skill in the artand are found in publically available databases, for example, thoseprovided by National Center for Biotechnology Information, U.S. NationalLibrary of Medicine8600 Rockville Pike, Bethesda Md., 20894USA, such asGENE (URL: http://www.ncbi.nlm.nih.gov/gene) or Office of Biological andEnvironmental Research of the U.S. Department of Energy Office ofScience, Human Genome Project Information (URL:http://genomics.energy.gov/).

The drug combination therapy involves administering each of the drugs inthe combination therapy in an amount sufficient to provide an observableimprovement over the baseline clinically observable signs and symptomsof the disorder treated with the combination. The drugs may be givenseparately (in a chronologically staggered manner, especially asequence-specific manner) in such time intervals that they prefer, suchthat in the patient shows a (preferably synergistic) interaction (jointtherapeutic effect), in particular wherein resistance to treatment withthe B-Raf inhibitor is overcome or reduced in the patient.

The term “pharmaceutically effective amount” or “clinically effectiveamount” or “therapeutically effective amount” of a combination oftherapeutic agents is an amount sufficient to provide an observableimprovement over the baseline clinically observable signs and symptomsof the disorder treated with the combination.

The general terms used herein are defined with the following meanings,unless explicitly stated otherwise:

The terms “comprising” and “including” are used herein in theiropen-ended and non-limiting sense unless otherwise noted.

The terms “a” and “an” and “the” and similar references in the contextof describing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Where the plural form is used for compounds, salts, and the like, thisis taken to mean also a single compound, salt, or the like.

The term “combination”, “therapeutic combination” “combination therapy”or “pharmaceutical combination”, as used herein, defines either a fixedcombination in one dosage unit form or a kit of parts or instructionsfor the combined administration where the B-Raf inhibitor and the secondinhibitor may be administered independently at the same time orseparately within time intervals that allow that the combinationpartners show a cooperative, e.g., synergistic, effect.

The term “pharmaceutical composition” is defined herein to refer to amixture or solution containing at least one therapeutic agent to beadministered to a subject, e.g., a mammal or human, in order to preventor treat a particular disease or condition affecting the mammal.

The term “pharmaceutically acceptable” is defined herein to refer tothose compounds, materials, compositions and/or dosage forms, which are,within the scope of sound medical judgment, suitable for contact withthe tissues a subject, e.g., a mammal or human, without excessivetoxicity, irritation allergic response and other problem complicationscommensurate with a reasonable benefit/risk ratio.

A “pharmaceutically acceptable salt”, as used herein, unless otherwiseindicated, includes salts of acidic and basic groups which may bepresent in the compounds of the present invention. The compounds of thepresent invention that are basic in nature are capable of forming a widevariety of salts with various inorganic and organic acids. The acidsthat may be used to prepare pharmaceutically acceptable acid additionsalts of such basic compounds of the present invention are those thatform non-toxic acid addition salts, i.e., salts containingpharmaceutically acceptable anions, such as the acetate, benzoate,bromide, chloride, citrate, fumarate, hydrobromide, hydrochloride,iodide, lactate, maleate, mandelate, nitrate, oxalate, salicylate,succinate, and tartrate salts. Unless otherwise specified, thetherapeutic agents used in the inventive methods are administered infree form or as a pharmaceutically salt.

The term “a combined preparation” is defined herein to refer toespecially a “kit of parts” in the sense that the combination partners(a) and (b) as defined above can be dosed independently or by use ofdifferent fixed combinations with distinguished amounts of thecombination partners (a) and (b), i.e., simultaneously or at differenttime points. The parts of the kit of parts can then e.g., beadministered simultaneously or chronologically staggered, that is atdifferent time points and with equal or different time intervals for anypart of the kit of parts. The ratio of the total amounts of thecombination partner (a) to the combination partner (b) to beadministered in the combined preparation can be varied, e.g., in orderto cope with the needs of a patient sub-population to be treated or theneeds of the single patient.

The term “co-administration” “combination therapy” or “combinedadministration” as used herein is defined to encompass theadministration of the selected therapeutic agents to a single patient,and are intended to include treatment regimens in which the agents arenot necessarily administered by the same route of administration or atthe same time.

The term “treating” or “treatment” as used herein comprises a treatmentrelieving, reducing or alleviating at least one symptom in a subject oreffecting a delay of progression of a disease. For example, treatmentcan be the diminishment of one or several symptoms of a disorder orcomplete eradication of a disorder, such as cancer. Within the meaningof the present invention, the term “treat” also denotes to arrest, delaythe onset (i.e., the period prior to clinical manifestation of adisease) and/or reduce the risk of developing or worsening a disease.The term “protect” is used herein to mean prevent, delay or treat, orall, as appropriate, development or continuance or aggravation of adisease in a subject.

The term “subject” or “patient” as used herein refers particularly to ahuman, e.g., a human suffering from, at risk of suffering from, orpotentially capable of suffering from the proliferative disease.However, it is not intended to exclude the treatment of mammals, e.g.,dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits rats andtransgenic non-human animals.

The term about” or “approximately” shall have the meaning of within 10%,more preferably within 5%, of a given value or range.

Thus, the present invention relates to a method for treating a patientsuffering from a proliferative disease characterized by a mutation inB-Raf, particularly a V600 mutation in B-Raf, very particularly amelanoma characterized by a V600 mutation in B-Raf, which comprises:

(a) obtaining a tumor sample from the patient and testing for a geneticalteration in a gene selected from the group comprising BRAF, CRAF,CCND1, CDK4, HER2, IGF-1R, cMET, FGFR1, FGFR2, FGFR3 EGFR, MAP2K1,MAP2K2, NRAS, KRAS HRAS, PTEN, PIK3CA, and P16.

(b) administering a drug combination therapy comprising a B-Rafinhibitor and a second inhibitor, which second inhibitor is selectedbased on genetic alterations discovered in the tumor sample inaccordance with Table 1, particularly wherein,

-   -   (i) the second inhibitor is a Mek 1/2 inhibitor when the tumor        sample has a genetic alteration in BRAF, CRAF, MAP2K1, MAPK2,        NRAS, KRAS HRAS or EGFR, or    -   (ii) the second inhibitor is a CDK 4 inhibitor when the tumor        sample has a genetic alteration in CCND1, CDK4 or P16, or    -   (iii) the second inhibitor is a PI3 Kinase inhibitor when the        tumor sample has a genetic alteration in HER2, IGF-1R, PTEN or        PIK3CA, or    -   (iv) the second inhibitor is a c-Met receptor tyrosine kinase        inhibitor when the tumor sample has a genetic alteration in        cMET,    -   (v) the second inhibitor is a FGFR kinase inhibitor when the        tumor sample has a genetic alteration in FGFR1, FGFR2 or FGFR3.

Thus, the present invention further relates to a method for treating apatient suffering from a proliferative disease characterized by amutation in B-Raf, particularly a V600 mutation in B-Raf, veryparticularly a melanoma characterized by a V600 mutation in B-Raf, whichcomprises:

(a) obtaining a tumor sample from the patient and detecting a geneticalteration in a gene selected from the group comprising BRAF, CRAF,MAP2K1, MAPK2, NRAS, KRAS HRAS or EGFR.

(b) administering a drug combination therapy to the patient comprisingthe B-Raf inhibitor and a second inhibitor which is a Mek 1/2 inhibitor.

The present invention also relates to a method for treating a patientsuffering from a proliferative disease characterized by a mutation inB-Raf, particularly a V600 mutation in B-Raf, very particularly amelanoma characterized by a V600 mutation in B-Raf, which comprises:

(b) obtaining a tumor sample from the patient and detecting a geneticalteration in a gene selected from the group comprising CCND1, CDK4 orP16.

(d) administering a drug combination therapy to the patient comprisingthe B-Raf inhibitor and a second inhibitor which is a CDK 4 inhibitor.

The present invention relates to a method for treating a patientsuffering from a proliferative disease characterized by a mutation inB-Raf, particularly a V600 mutation in B-Raf, very particularly amelanoma characterized by a V600 mutation in B-Raf, which comprises:

(a) obtaining a tumor sample from the patient after disease progressionand detecting a genetic alteration in a gene selected from the groupcomprising HER2, IGF-1R, PTEN or PIK3CA,

(b) administering a drug combination therapy to the patient comprisingthe B-Raf inhibitor and a second inhibitor which is a PI3 Kinaseinhibitor.

The present invention relates to a method for treating a patientsuffering from a proliferative disease characterized by a mutation inB-Raf, particularly a V600 mutation in B-Raf, very particularly amelanoma characterized by a V600 mutation in B-Raf, which comprises:

(a) obtaining a tumor sample and detecting a genetic alteration in agene selected from the group comprising cMET,

(b) administering a drug combination therapy to the patient comprisingthe B-Raf inhibitor and a second inhibitor which is a c-Met receptortyrosine kinase inhibitor.

The present invention relates to a method for treating a patientsuffering from a proliferative disease characterized by a mutation inB-Raf, particularly a V600 mutation in B-Raf, very particularly amelanoma characterized by a V600 mutation in B-Raf, which comprises:

(a) obtaining a tumor sample from the patient and detecting a geneticalteration in a gene selected from the group comprising FGFR1, FGFR2 orFGFR3

(b) administering a drug combination therapy to the patient comprisingthe B-Raf inhibitor and a second inhibitor which is a FGFR kinaseinhibitor.

In an important embodiment of the present invention, the patient hasbeen treated previously with B-Raf inhibitor monotherapy. Particularly,the patient is treated with B-Raf inhibitor monotherapy until diseaseprogression followed by a drug combination therapy determined inaccordance with Table 1.

In a preferred embodiment, the B-Raf inhibitor is administeredcontinuously as a monotherapy until disease progression or initiation ofthe drug combination therapy and the continuous administration iscontinued during treatment with the drug combination therapy.

In another embodiment, the B-Raf inhibitor is administered on anintermittent dosing schedule, which means that that the B-Raf inhibitoris administered for a period of time followed by a period of timewherein treatment with the B-Raf inhibitor is withheld. For example, theRaf inhibitor is administered daily for a period of 3 or 4 weeksfollowed by a period of 1 or 2 weeks without treatment and the cycle isrepeated.

Disease progression is evaluated by appropriate clinical criteria, suchas the RECIST criteria. RECIST (Response Evaluation Criteria In SolidTumors) is a set of published rules that define when cancer patientsimprove (“respond”), stay the same (“stable”) or worsen (“progression”)during treatments. The original criteria were published in February 2000by an international collaboration including the European Organizationfor Research and Treatment of Cancer (EORTC), National Cancer Institute(NCI) of the United States and the National Cancer Institute of CanadaClinical Trials Group. RECIST 1.1, published in January 2009, is anupdate to the original criteria. See, Eur. J. Cancer, 45, (2009)228-247.

A mechanism for disease progression is determined by comparison of thegenetic alterations present in a patient's tumor at the time of relapse,for example, versus pre-treatment. The genetic alterations can resultfrom amplification of a gene, mutations in a gene or loss of the gene'sactivity. The genetic alterations are determined by methods known in theart, typically by known sequencing methods. In a preferred embodiment,genes selected from the group consisting of B-Raf, C-Raf, CCND1, CDK4,HER2, IGF-1R, cMET, FGFR1, FGFR2, FGFR3 EGFR, MAP2K1, MAP2K2, NRAS, KRASHRAS, PTEN, PIK3CA, and P16 in tumor samples taken at the time ofrelapse versus pre-treatment are compared.

Thus, the present invention also relates to testing a tumor sampleobtained from a patient suffering from a proliferative diseasecharacterized by a mutation in B-Raf, particularly a V600 mutation inB-Raf, very particularly a melanoma characterized by a V600 mutation inB-Raf, for genetic alterations in a panel of genes comprising B-Raf,C-Raf, CCND1, CDK4, HER2, IGF-1R, cMET, FGFR1, FGFR2, FGFR3 EGFR,MAP2K1, MAP2K2, NRAS, KRAS HRAS, PTEN, PIK3CA, and P16 in order todetermine a mechanism of disease progression after treatment with aB-Raf inhibitor.

The present invention also relates to a diagnostic method for selectinga second inhibitor to be combined with a B-Raf inhibitor wherein a tumorsample is tested for genetic alterations one of more genes selected fromB-Raf, C-Raf, CCND1, CDK4, HER2, IGF-1R, cMET, FGFR1, FGFR2, FGFR3 EGFR,MAP2K1, MAP2K2, NRAS, KRAS HRAS, PTEN, PIK3CA, and P16. The secondinhibitor is selected in accordance with Table 1. Preferably, the secondinhibitor is selected to overcome resistance to treatment with the B-Rafinhibitor.

The present invention also relates a gene chip useful for detecting forgenetic alterations in one of more genes selected from B-Raf, C-Raf,CCND1, CDK4, HER2, IGF-1R, cMET, FGFR1, FGFR2, FGFR3 EGFR, MAP2K1,MAP2K2, NRAS, KRAS HRAS, PTEN, PIK3CA, and P16, or which comprises allor a subset of the aforementioned genes. The gene chip is useful fordetermining a mechanism of resistance to treatment with a B-Rafinhibitor and for selecting a second inhibitor to be used in drugcombination therapy which overcomes that resistance.

A particular embodiment of the present invention is a method fortreating a patient suffering from a proliferative disease characterizedby a mutation in B-Raf, particularly a V600 mutation in B-Raf, veryparticularly a melanoma characterized by a V600 mutation in B-Raf, whichcomprises:

(a) administering a therapeutically effective amount of a B-Rafinhibitor to the patient until the patient exhibits disease progression,

(b) obtaining a tumor sample from the patient after disease progressionand testing for a genetic alteration in one or more genes selected fromthe group consisting of BRAF, CRAF, CCND1, CDK4, HER2, IGF-1R, cMET,FGFR1, FGFR2, FGFR3 EGFR, MAP2K1, MAP2K2, NRAS, KRAS HRAS, PTEN, PIK3CA,and P16, and(c) administering a drug combination therapy comprising the B-Rafinhibitor and a second inhibitor, which second inhibitor is selectedbased on genetic alterations found in the tumor sample, wherein,

-   -   (i) the second inhibitor is a Mek 1/2 inhibitor when the genetic        alteration is in BRAF, CRAF, MAP2K1, MAPK2, NRAS, KRAS HRAS or        EGFR, or    -   (ii) the second inhibitor is a CDK 4 inhibitor when the genetic        alteration is in CCND1, CDK4 or P16, or    -   (iii) the second inhibitor is a PI3 Kinase inhibitor when the        genetic alteration is in HER2, IGF-1R, PTEN or PIK3CA, or    -   (iv) the second inhibitor is a c-Met receptor tyrosine kinase        inhibitor when the genetic alteration is in cMET, or    -   (v) the second inhibitor is a FGFR kinase inhibitor when the        genetic alteration is in FGFR1, FGFR2 or FGFR3.

A preferred B-Raf inhibitor useful in the present invention is aCompound of Formula (I)

The Compound of formula (I) and its utility as a B-Raf inhibitor aredisclosed in WO 2011/025927.

Thus, the present invention more particularly relates to a method fortreating a patient suffering from a proliferative disease characterizedby a mutation in B-Raf, particularly a V600 mutation in B-Raf, veryparticularly a melanoma characterized by a V600 mutation in B-Raf, whichcomprises:

(a) obtaining a tumor sample from the patient and testing for a geneticalteration in one or more genes selected from the group consisting ofBRAF, CRAF, CCND1, CDK4, HER2, IGF-1R, cMET, FGFR1, FGFR2, FGFR3 EGFR,MAP2K1, MAP2K2, NRAS, KRAS HRAS, PTEN, PIK3CA, and P16, and(b) administering a drug combination therapy comprising a B-Rafinhibitor of the formula (I)

or a pharmaceutically acceptable salt thereof, and a second inhibitor,which second inhibitor is selected based on genetic alterationsdiscovered in the tumor sample in accordance with Table 1, particularlywherein,

-   -   (i) the second inhibitor is a Mek 1/2 inhibitor when the tumor        sample has a genetic alteration in BRAF, CRAF, MAP2K1, MAPK2,        NRAS, KRAS HRAS or EGFR, or when no genetic alteration is found        in step (b), or    -   (ii) the second inhibitor is a CDK 4 inhibitor when the tumor        sample has a genetic alteration in CCND1, CDK4 or P16, or    -   (iii) the second inhibitor is a PI3 Kinase inhibitor when the        tumor sample has a genetic alteration in HER2, IGF-1R, PTEN or        PIK3CA, or    -   (iv) the second inhibitor is a c-Met receptor tyrosine kinase        inhibitor when the tumor sample has a genetic alteration in        cMET, or    -   (v) the second inhibitor is a FGFR kinase inhibitor when the        tumor sample has a genetic alteration in FGFR1, FGFR2 or FGFR3.

A more specific embodiment of the present invention includes providingmonotherapy with the B-Raf inhibitor of Formula (I) prior to the drugcombination therapy. Thus, the present invention further relates to amethod for treating a patient suffering from a proliferative diseasecharacterized by a mutation in B-Raf, particularly a V600 mutation inB-Raf, very particularly a melanoma characterized by a V600 mutation inB-Raf, which comprises:

(a) administering to the patient a therapeutically effective amount of aB-Raf inhibitor of the formula (I)

or a pharmaceutically acceptable salt thereof, until the patientexhibits disease progression,(b) obtaining a tumor sample from the patient after disease progressionand testing for a genetic alteration in one or more genes selected fromthe group consisting of B-Raf, C-Raf, CCND1, CDK4, HER2, IGF-1R, cMET,FGFR1, FGFR2, FGFR3 EGFR, MAP2K1, MAP2K2, NRAS, KRAS HRAS, PTEN, PIK3CA,and P16,(c) administering a drug combination therapy comprising the B-Rafinhibitor and a second inhibitor, which second inhibitor is selectedbased on the genetic alteration discovered in the tumor sample inaccordance with Table 1, particularly wherein,

-   -   (i) the second inhibitor is a Mek 1/2 inhibitor when the        mechanism of disease progression is characterized by a genetic        alteration in BRAF, CRAF, MAP2K1, MAPK2, NRAS, KRAS HRAS or        EGFR, or when no genetic alteration is found in step (b), or    -   (ii) the second inhibitor is a CDK 4 inhibitor when the        mechanism of disease progression is characterized by a genetic        alteration in CCND1, CDK4 or P16, or    -   (iii) the second inhibitor is a PI3 Kinase inhibitor when the        mechanism of disease progression is characterized by a genetic        alteration in HER2, IGF-1R, PTEN or PIK3CA, or    -   (iv) the second inhibitor is a c-Met receptor tyrosine kinase        inhibitor when the mechanism of disease progression is        characterized by a genetic alteration in cMET, or    -   (v) the second inhibitor is a FGFR kinase inhibitor when the        mechanism of disease progression is characterized by a genetic        alteration in FGFR1, FGFR2 or FGFR3.

The Compound of Formula (I) may be administered continuously or on anintermittent dosing schedule in steps (a) and (c). It is preferablyadministered continuously.

In each of the aforementioned methods, preferred embodiments especiallyinclude those wherein the proliferative disease is characterized by aV600 mutation in B-Raf, for example a V600E mutation. Proliferativediseases frequently characterized by such a mutation include melanoma,colorectal cancer, thyroid cancer, particularly papillary thyroidcancer, astrocytomas, pancreatic cancer, and neurofibromatosis.Preferably, the proliferative disease is melanoma or colorectal cancercharacterized by a V600 mutation in B-Raf, for example a V600E, V600K orV600G mutation. The present invention especially relates to such amethod wherein the proliferative disease is melanoma characterized by aV600 mutation in B-Raf, for example a V600E, V600K or V600G mutation.

Appropriate Mek 1/2 inhibitors for use in the present method are knownin the art. Mek 1/2 inhibitors useful in the present invention includePD325901, PD-181461, ARRY142886/AZD6244, ARRY-509, XL518, JTP-74057,AS-701255, AS-701173, AZD8330, ARRY162, ARRY300, RDEA436, E6201,RO4987655/R-7167, GSK1120212 or AS703026.

In an important embodiment, the Mek 1/2 inhibitors include compoundsdescribed in WO03/077914, which is here incorporated by reference in itsentirety, in particular a compound of formula (II) or (III).

or pharmaceutically acceptable salts thereof, (hereinafter referred toas Compounds A and B, respectively) and the compounds described inWO05/051906, WO05/023251, WO03/077855, US20050049419, and U.S. Pat. No.7,235,537, which are here incorporated by reference in their entirety,covering N3-alkylated benzimidazoles and other similar heterocyclicderivatives as Mek 1/2 inhibitors for the treatment of proliferativediseases.

CDK 4 inhibitors are known in the art and include flavopiridol,P1446A-05, LEE011, AT7519, BMS265246, LY2835219 and PD-0332991. In aparticular embodiment of the present invention, the CDK 4 inhibitor is acompound disclosed in WO2007/140222 or WO 20210/020675, which are hereincorporated by reference in their entirety. In a particular embodiment,the CDK 4 inhibitor is a compound of the formula (IV)

or a pharmaceutically acceptable salt thereof, hereinafter referred toas compound C.

PI3 Kinase inhibitors are known in the art and include perifosine,CAL-101, PX-866, BEZ235, SF1126, INK1117, GDC-0941, BKM120, XL147,XL765, Palomid529, GSK1059615, Zstk474, PTW33597, IC87114, TG100-115,CAL283, PI-103, BYL719, GNE-477, CUDC-907, and AEZS-136.

WO2006/122806, which is here incorporated by reference in its entirety,describes imidazoquinoline derivatives having PI3-kinase inhibitoryactivity. A very preferred compound of the present invention is2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrileand its monotosylate salt (COMPOUND D). The synthesis of2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrileis for instance described in WO2006/122806 as Examples 7 and 152-3.Another very preferred compound of the present invention is8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one(COMPOUND E). The synthesis of8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-oneis for instance described in WO2006/122806 as Example 86. WO07/084786describes pyrimidine derivatives having PI3 Kinase inhibitory activity.A very preferred compound of the present invention is5-(2,6-di-morpholin-4-yl-pyrimidin-4-yl)-4-trifluoromethyl-pyridin-2-ylamine(COMPOUND F). The synthesis of5-(2,6-di-morpholin-4-yl-pyrimidin-4-yl)-4-trifluoromethyl-pyridin-2-ylamineis described in WO07/084786 as Example 10. Another preferred compoundhaving PI3-kinase inhibitory activity is(S)-Pyrrolidine-1,2-dicarboxylic acid 2-amide1-({4-methyl-5-[2-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl}-amide)(Compound X).

c-Met receptor tyrosine kinase inhibitors are known in the art andinclude crizotinib, PHA-665752, SU11274, PF-04217903, foretinib, SGX523,JNJ-38877605, GSK1363089, AMG208, and INCB28060. In a particularembodiment, the c-Met receptor tyrosine kinase inhibitor is a compoundof the formula IV

or a pharmaceutically acceptable salt thereof, (hereinafter Compound G).

FGFR kinase inhibitors used according to the present method arepreferably selective and ATP competitive pan FGFR kinase inhibitorincluding AZD4547 and BGJ398. In a particular embodiment, the FGFRkinase inhibitor is an aryl-pyrimidyl-urea derivative disclosed inWO2006/000420, particularly a compound of the formula (V)

or a pharmaceutically acceptable salt thereof (hereinafter Compound H).

Particular preference is given to embodiments of the inventive methodswherein the Mek 1/2 inhibitor is Compound A or Compound B, particularlycompound B, the CDK 4 inhibitor is Compound C, the PI3 Kinase inhibitoris Compound D, Compound E, Compound F or Compound X, particularlyCompound F, the c-Met receptor tyrosine kinase inhibitor is compound Gand wherein the FGFR kinase inhibitor is Compound H, or apharmaceutically acceptable salt of the aforementioned compounds.

The B-Raf inhibitor of Formula (I) is administered at a dose of 150 to600 per day, preferably 400 to 600 per day, particularly 450 or 600mg/day. As the second inhibitor of the drug combination therapy CompoundB is administered at a dose of 15 to 60 mg BID, preferably 45 mg BID,Compound C is administered at a dose of 100 to 900 mg/day, preferably200 to 900 mg/day, for example, 200, 400, 700 or 900 mg/day, Compound Fis administered at a dose of 30 to 100 mg/day, preferably 60 to 100mg/day or 60 to 80 mg/day, Compound G is administered at a dose of 50 to300 mg BID, preferably 100 to 300 mg BID, for example, 100, 150, 200,250 or 300 mg BID, or Compound H is administered at a dose of 25 to 125mg/day, for example, 75, 100 or 125 mg/day.

In an important embodiment of the aforementioned methods, the geneticalteration in BRAF discovered in the tumor sample is other than a V600mutation.

The present invention further relates to therapeutic combinationscomprising a B-Raf inhibitor, preferably a B-Raf inhibitor of Formula(I) and a second inhibitor selected from the group consisting of a PI3Kinase inhibitor, a c-Met receptor tyrosine kinase inhibitor and a FGFRkinase inhibitor for separate, simultaneous or sequentialadministration. More particularly, the therapeutic combination comprisesa B-Raf inhibitor of Formula (I) and a second inhibitor which is a PI3Kinase inhibitor selected from the group consisting of Compound D,Compound E, Compound F, and Compound X, or a pharmaceutically acceptablesalt thereof; or the therapeutic combination comprises a B-Raf inhibitorof Formula (I) and a second inhibitor which is a c-Met inhibitorselected from Compound G, or a pharmaceutically acceptable salt thereof;or the therapeutic combination comprises a B-Raf inhibitor of Formula(I) and a second inhibitor which is a FGFR kinase inhibitor selectedfrom Compound H, or a pharmaceutically acceptable salt thereof, forseparate, simultaneous or sequential administration. Hereinafter, suchtherapeutic combinations are referred to as a COMBINATION OF THEINVENTION.

The present invention further relates to a method for treating a patientsuffering from a proliferative disease characterized by a mutation inB-Raf, for example, melanoma characterized by a V600 mutation in B-Raf,which comprises, administering to the patient a therapeuticallyeffective amount of a combination comprising a B-Raf inhibitor,preferably a B-Raf inhibitor of Formula (I) and a second inhibitorselected from the group consisting of a PI3 Kinase inhibitor, a c-Metreceptor tyrosine kinase inhibitor and a FGFR kinase inhibitor. Moreparticularly, the present invention relates to a method for treating apatient suffering from a proliferative disease characterized by amutation in B-Raf, such as a V600 mutation, for example, melanomacharacterized by a V600 mutation in B-Raf, which comprises,administering to the patient a therapeutically effective amount of aCOMBINATION OF THE INVENTION. Preferably, these inhibitors areadministered at therapeutically effective dosages which, when combined,provide a beneficial effect. The administration may be separate,simultaneous or sequential.

The present invention also pertains to a COMBINATION OF THE INVENTIONfor use in the preparation of a pharmaceutical composition or medicamentfor the treatment or prevention of a proliferative disease, particularlya proliferative disease characterized by a mutation in B-Raf, especiallya V600 mutation in B-Raf, for example, melanoma characterized by a V600mutation in B-Raf, in a patient in need thereof.

The present invention further provides a commercial package comprisingas therapeutic agents a COMBINATION OF THE INVENTION, together withinstructions for simultaneous, separate or sequential administrationthereof for use in the delay of progression or treatment of aproliferative disease.

The administration of a COMBINATION OF THE INVENTION may result not onlyin a beneficial effect, e.g. a synergistic therapeutic effect, e.g. withregard to alleviating, delaying progression of or inhibiting thesymptoms, but also in further surprising beneficial effects, e.g. fewerside-effects, more durable response, an improved quality of life or adecreased morbidity, compared with a monotherapy applying only one ofthe pharmaceutically therapeutic agents used in the combination of theinvention.

The following examples are intended to illustrate, but not limit, theinvention.

EXAMPLE 1

During the first part of the study, patients are treated with the B-Rafinhibitor of Formula (I) as a single agent at the Recommended Phase IIDose of 450 mg/day. The B-Raf inhibitor of Formula (I) is administeredorally formulated as an encapsulated solid dispersion.

In the second part of the study, patients will be treated with the B-Rafinhibitor of Formula (I) in combination with a second targeted agent(i.e. the B-Raf inhibitor of Formula (I)+Compound B, the B-Raf inhibitorof Formula (I)+Compound F, the B-Raf inhibitor of Formula (I)+CompoundH, the B-Raf inhibitor of Formula (I)+Compound G, or the B-Raf inhibitorof Formula (I)+Compound C). The dose escalation in each combination armwill be guided by a Bayesian logistic regression model (BLRM) in orderto establish the MTD/RP2D of each combinations unless it has beenpreviously determined in a separate combination trial. The open-labeldose escalation study design using a BLRM is a well-established methodto estimate the MTD(s) and/or RP2D(s) in cancer patients. The adaptiveBLRM will be guided by the escalation with overdose control (EWOC)principle to control the risk of DLT in future patients on study.Intra-patient dose escalation will be permitted after the first cyclefor patients who have not experienced a DLT.

Intra-patient dose escalation will be guided by the BLRM with a modifiedEWOC criteria that reflects individual patient tolerability. The use ofBayesian response adaptive models for small datasets has been acceptedby EMEA and its development and appropriate use is one aspect of theFDA's Critical Path Initiative.

Rationale for Choice of Combination Drugs

Data from pre-clinical and clinical studies suggest that bysimultaneous, dual, vertical pathway inhibition of the RAF/MEK/ERKsignaling pathway with the B-Raf inhibitor of Formula (I) and Compound Bcombination could lead to increased clinical efficacy and possiblyovercome early resistance to either single agent in patients with BRAFV600-dependent advanced melanoma. Moreover, other mechanisms thatreactivate MAPK signaling or activate alternate pathways such asPI3K/AKT signaling pathway may play a role in primary and/or acquiredresistance to BRAF inhibitors. Thus the antitumor activity of the B-Rafinhibitor of Formula (I) in combination with selected agent Compound F,Compound H, Compound G, and Compound C that target PI3K, c-met, FGFR andCDK4/6 kinase respectively, will also be assessed in addition to theB-Raf inhibitor of Formula (I)+Compound B. The selection of the B-Rafinhibitor of Formula (I) combination given to an individual patient willbe based on the genetic alteration(s) identified in this patient's tumorsample upon the B-Raf inhibitor of Formula (I) progression (see table1).

Description of Study Design

This is a multicenter, open-label, phase II study which will enrollapproximately 100 patients with BRAF mutant locally advanced ormetastatic melanoma, and consists of two treatment parts.

In the first part, Part I, patients naïve to a selective BRAF inhibitorwill be treated with the B-Raf inhibitor of Formula (I) single agent atthe RP2D of 450 mg/day until disease progression (as defined per RECISTv1.1). At the time of disease progression, the tumor will be biopsiedand analyzed for a selected panel of genes (Table 1).

The patients relapsing in Part I of the study will continue to receivethe B-Raf inhibitor of Formula (I) single agent, during the expectedturn-around time for the molecular analysis of the resistance biopsy,until the appropriate the B-Raf inhibitor of Formula (I) rationalcombination can be identified and initiated.

Based upon genetic alterations identified from the tumor biopsy atrelapse, cohorts of patients will enter in the second part of the study,Part II, for a tailored combination treatment of the B-Raf inhibitor ofFormula (I) plus a second targeted agent. There will be 5 armscorresponding to the 5 combination treatment studied: the B-Rafinhibitor of Formula (I)+Compound B, the B-Raf inhibitor of Formula(I)+Compound F, the B-Raf inhibitor of Formula (I)+Compound H, the B-Rafinhibitor of Formula (I)+Compound G, and the B-Raf inhibitor of Formula(I)+Compound C. The selection of the second agent will be definedfollowing Table 1 criteria. It is anticipated that more than half of thepatients enrolled will receive a combination treatment of the B-Rafinhibitor of Formula (I) plus Compound B after progression on the B-Rafinhibitor of Formula (I).

Non-naïve patients for BRAF inhibitor treatment who are relapsing in aprior study in which patients with BRAF V600 mutant melanoma weretreated with the B-Raf inhibitor of Formula (I) single agent can beenrolled after progression on the B-Raf inhibitor of Formula (I) singleagent in Part II. For these patients, the analysis results of the freshtumor biopsy collected at the End of Treatment visit from the previoustrial will be used for combination treatment assignment in Part II.

The patients relapsing in other the B-Raf inhibitor of Formula (I)single agent studies (e.g IIT), will be discontinued from the the B-Rafinhibitor of Formula (I) treatment after progression and will stopreceiving the B-Raf inhibitor of Formula (I) single agent, until theycan be assigned to a rational combination treatment in part II of thestudy.

Progressive disease of those patients is deemed to be confirmed from theprevious study and will be used as baseline tumor evaluation for Part IIof the study if a time interval before the CT at progression and thestart of study treatment within this study is no longer than 28 days.

All patients will start the rational combination at the defined dualcombination MTD/RP2D, or if the dual combination MTD/RP2D has not beenpreviously determined, at the RP2D of 450 mg/day for the B-Raf inhibitorof Formula (I) (or the highest last-dose tolerated by the patient) incombination with the second agent at a starting dose allowed by theBayesian logistic regression model. The rational combination treatmentpart will continue with the possibility of ascending dose of the secondagent until a MTD/RP2D of the combination has been established. Intrapatient dose escalation of the second agent guided by a BLRM will beallowed under pre-defined conditions for patients who tolerated thecombination at a given dose for at least one cycle.

Combination treatment will be administered in 21-day cycles untildisease progression, where the tumor assessments during combinationtreatment will be compared to a recalculated baseline (i.e., the resultof tumor evaluation leading to PD assessment on the B-Raf inhibitor ofFormula (I) single agent treatment either in part I or in previousstudy).

Molecular Pre-Screening

To enter the screening phase of the study, patients must have writtendocumentation of BRAF V600 mutation, which should be obtained locally ona fresh tumor biopsy (preferred) or the most recent archival tumorsample available. However, patients for whom molecular status is notknown at the time of consideration for enrollment in this study and whohave a tumor which is not routinely screened for a BRAF mutation at alocal laboratory and for whom fresh tumor collection is required, willsign a molecular pre-screening Informed Consent allowing for thecollection of fresh tumor sample for local assessment of the mutationalstatus. Only once the BRAF V600 mutational status is known ordetermined, the patient is allowed to sign the main Study InformedConsent Form and start screening.

Screening

Once the BRAF V600 mutational status is known or determined, the patientis allowed to sign the Main Study Informed Consent Form and startscreening. All screening evaluations are required to be performed beforeadministration of study treatment.

Treatment Period

There will be two treatment parts: Part I and Part II:

Part I=the single agent treatment phase and will begin on Cycle 1 Day 1until initiation of combination treatment.

Part II=the combination treatment, should be initiated once the geneticalterations from tumor biopsy collection at the time of relapse areknown.

Study treatments will be administered during 21-day cycles and willcontinue until disease progression (on dual combination treatment),unacceptable toxicity, withdrawal of informed consent, or death.

Patient Population

The study will be conducted in adult patients with locally advanced ormetastatic melanoma harboring a confirmed BRAF V600 mutation,

Patients enrolled in the first part of the trial (Part I) must be naïveto a selective BRAF inhibitor.

Patients previously treated with the B-Raf inhibitor of Formula (I)single agent can be enrolled directly in Part II if a tumor biopsy iscollected at the time of relapse.

Patients enrolled in this study are not permitted to participate inparallel investigational drug or device studies. Additionally, patientswho have completed the study must not be re-enrolled for a second courseof treatment.

The investigator or designee must ensure that only patients who meet allthe following inclusion and none of the exclusion criteria are offeredtreatment in the study.

Inclusion Criteria

Patients naïve for the B-Raf inhibitor of Formula (I) (eligible for PartI).

Patients eligible for inclusion in the study have to meet all of thefollowing criteria:

Age ≧18 years at the start of dosing

Able to understand and voluntarily sign the informed consent form, andability to comply with the study visit schedule and other protocolrequirements. Written informed consent must be obtained prior to screenprocedures

Histologically confirmed diagnosis of unresectable stage III ormetastatic melanoma (stage IIIC to IV per American Joint Committee onCancer [AJCC]).

Written documentation of BRAF V600 mutation,

Fresh tumor biopsy at baseline, and patient agrees for a mandatorybiopsy at the time of relapse, if not medically contraindicated.

Evidence of measurable disease, as determined by RECIST v1.1.

Note: Lesions in areas of prior radiotherapy or other locoregionaltherapies (e.g., percutaneous ablation) should not be consideredmeasurable, unless lesion progression has been documented since thetherapy.

Life expectancy ≧3 months

World Health Organization (WHO) Performance Status ≦2.

Negative serum pregnancy test within 72 hours prior to the first dose ofstudy treatment in all women of childbearing potential.

A mandatory fresh biopsy at relapse after the B-Raf inhibitor of Formula(I) single agent treatment must be available.

The patients of in other single agent studies of the B-Raf inhibitor ofFormula (I) with documented progressive disease could join the Part IIaccording the resistance profile results which will determine thecombination arm assignment for treatment.

Progressive disease of those patients has to be confirmed from theprevious study with a tumor evaluation assessment. If the time intervalbetween the tumor evaluation documenting the disease progression and thefirst dose of the combination treatment is more than 4 weeks (28 days),a new tumor evaluation should be performed. The biopsy performed at theEnd of Treatment visit of the previous study and characterized through acomprehensive genomic analysis will be required for the assignment ofthe combination treatment.

Exclusion Criteria

Patients eligible for this study must not meet any of the followingcriteria:

Enrollment in Part I (the B-Raf inhibitor of Formula (I) single agenttreatment):

Previous treatment with RAF-inhibitor

Symptomatic or Untreated Leptomeningeal Disease

Symptomatic brain metastases. Patients previously treated or untreatedfor these conditions that are asymptomatic in the absence ofcorticosteroid therapy are allowed to enroll. Brain metastasis must bestable at least three months with verification by imaging (e.g. brainMRI or CT completed at screening demonstrating no current evidence ofprogressive brain metastases). Patients are not permitted to receiveenzyme inducing anti-epileptic drugs.

Known Acute or Chronic Pancreatitis

Clinically significant cardiac disease including any of the following:

CHF requiring treatment (NYH grade ≧2), LVEF <45% as determined by MUGAscan or ECHO, or uncontrolled hypertension (please refer to WHO-ISHguidelines)

History or presence of clinically significant ventricular arrhythmias oratrial fibrillation

Clinically Significant Resting Bradycardia

Unstable angina pectoris ≦3 months prior to starting study drug

Acute Myocardial Infarction (AMI)≦3 months prior to starting study drug

QTcF >480 msec on screening ECGs

Patients with any of the following laboratory values at baseline:

Absolute neutrophil count (ANC) <1,500/mm3 [1.5×109/L]

Platelets <100,000/mm3 [100×109/L]

Hemoglobin <9.0 g/dL

Serum creatinine >1.5×ULN

Serum total bilirubin >1.5×ULN

AST/SGOT and ALT/SGPT >2.5×ULN, or >5×ULN if liver metastases arepresent

Impairment of gastrointestinal (GI) function or GI disease that maysignificantly alter the absorption of oral interventional drug (e.g.,ulcerative diseases, uncontrolled nausea, vomiting, diarrhea,malabsorption syndrome, small bowel resection).

Previous or concurrent malignancy. Exceptions: adequately treated basalcell or squamous cell skin cancer; in situ carcinoma of the cervix,treated curatively and without evidence of recurrence for at least 3years prior to study entry; or other solid tumor treated curatively, andwithout evidence of recurrence for at least 3 years prior to studyentry.

History of thromboembolic or cerebrovascular events within the last 6months, including transient ischemic attack, cerebrovascular accident,deep vein thrombosis, or pulmonary embolism.

Patients who have received radiation therapy (that includes >30% of thebone marrow reserve), chemotherapy, biological therapy (e.g.,antibodies) within ≦4 weeks (6 weeks for nitrosourea, mitomycin-C), orwho have been treated with continuous or intermittent small moleculetherapeutics or investigational agents within 5-half-lives of the agent(or ≦4 weeks when half-life is unknown) prior to starting study drug orwho have not recovered from the side effects of such therapy (exceptalopecia).

Patients who have undergone any major surgery within the last 2 weeksprior to starting study drug or who would not have fully recovered fromprevious surgery.

Known Human Immunodeficiency Virus (HIV) Infection.

Other severe, acute, or chronic medical or psychiatric condition orlaboratory abnormality that may increase the risk associated with studyparticipation or study drug administration or that may interfere withthe interpretation of study results and, in the judgment of theinvestigator, would make the patient inappropriate for the study.

Pregnant or nursing (lactating) women, where pregnancy is defined as thestate of a female after conception and until the termination ofgestation, confirmed by a positive hCG laboratory test (>5 mlU/mL).Women of child-bearing potential, defined as all women physiologicallycapable of becoming pregnant, are not allowed to participate in thisstudy UNLESS they are using highly effective methods of contraceptionthroughout the study and for 10 days after study drug discontinuation.

Post-menopausal women are allowed to participate in this study. Womenare considered post-menopausal and not of child bearing potential ifthey have had 12 months of natural (spontaneous) amenorrhea with anappropriate clinical profile (e.g. age appropriate, history of vasomotorsymptoms) or six months of spontaneous amenorrhea with serumFollicle-Stimulating Hormone (FSH) levels >40 mlU/mL or have hadsurgical bilateral oophorectomy (with or without hysterectomy) or tuballigation at least six weeks prior to screening. In the case ofoophorectomy alone, only when the reproductive status of the woman hasbeen confirmed by follow-up hormone level assessment is she considerednot of child bearing potential.

Sexually active males must use a condom during intercourse while takingthe drug and for 3 months after stopping treatment and should not fathera child in this period. A condom is required to be used also byvasectomized men in order to prevent delivery of the drug via seminalfluid.

Study Treatment

The investigational drugs to be used in this study are the B-Rafinhibitor of Formula (I), Compound B, Compound F, Compound H, CompoundG, and Compound C.

The study treatments are:

Part I: single agent the B-Raf inhibitor of Formula (I)

Part II: dual combinations

the B-Raf inhibitor of Formula (I) (QD) and Compound B (BID)

the B-Raf inhibitor of Formula (I) (QD) and Compound F (QD)

the B-Raf inhibitor of Formula (I) (QD) and Compound H (QD)

the B-Raf inhibitor of Formula (I) (QD) and Compound G (BID)

the B-Raf inhibitor of Formula (I) (QD) and Compound C (QD)

Dosing Regimens

TABLE 2 Dose and treatment schedule Pharmaceutical form and route ofStarting Dose Study treatments administration (21 days cycles) the B-Rafinhibitor of Capsule for oral use 450 mg/day, or highest Formula (I)tolerated dose Compound B Tablet for oral use 45 mg BID Compound FCapsule for oral use 60 mg Compound H Capsule for oral use 75 mgCompound G Capsule for oral use 150 mg BID Compound C Capsule for oraluse 200 mgInstructions for Administration of the B-Raf Inhibitor of Formula(I)+Second Agent

The B-Raf inhibitor of Formula (I), Compound F, Compound H and CompoundC will be administered orally on a daily schedule (QD) as a flat-fixeddose, and not by body weight or body surface area.

QD Dosing: Patients should be instructed to take the B-Raf inhibitor ofFormula (I) (and Compound F, Compound H or Compound C) capsules dailywith a large glass of water (˜250 ml) in the morning. On all doseadministrations patients should fast for 2 hours prior to and afterstudy drug intake. If the patient forgets to take the dose in themorning, then he/she should take the dose within 6 hrs after the misseddose. If more than 6 hours has passed, then the dose should be withheldthat day and the patient should continue treatment with the nextscheduled dose. If, for any reason, a breakfast was not consumed, thenthe patient should still take the scheduled morning dose with a glass ofwater. If this happens on days of full PK sampling, it should bedocumented.

Compound B and Compound G will be administered orally on a twice dailyschedule (BID) as a flat-fixed dose, and not by body weight or bodysurface area.

BID Dosing: The doses of Compound B, or Compound G, should be taken 12±2hours apart. Patients will be instructed to take doses daily with alarge glass of water (˜250 ml) in the morning and in the evening. Forthe B-Raf inhibitor of Formula (I) and Compound G combination, on alldose administrations patients should fast for 2 hours prior to and afterstudy drug intake. For the B-Raf inhibitor of Formula (I) and Compound Bcombination, on all morning dose administration days patients should noteat anything within 2 hours prior to study drug intake and refrain fromeating for 2 hours following the B-Raf inhibitor of Formula (I) andCompound B intake. On all evening dose administrations patients shouldfast for 1 hour prior to and after Compound B intake Note that bothdrugs (the B-Raf inhibitor of Formula (I)+Compound B, or Compound G)should be taken together in the morning and only the BID administereddrug (Compound B, or Compound G) should be taken in the evening.

Instructions for Administration on Days when a PK Sampling is Performed:

Pre-dose PK samples should be collected just prior to intake of dose.

At each visit, responsible site personnel will ensure that theappropriate dose of each study drug is administered and will provide thepatient with the correct amount of study drug(s) for subsequent dosing.Patients will be instructed to return unused study drugs to the site ateach visit.

Patients should be instructed to swallow the capsules/tablets whole andnot to chew or crush them.

Any doses that are skipped should not be replaced or made up during thenext scheduled dosing or on a subsequent day, whichever applies.

Patients must avoid consumption of grapefruit, pomegranates, starfruits, Seville oranges or products containing the juice of each duringthe entire study and preferably 7 days before the first dose of studymedications, due to potential CYP3A4 interaction with the studymedications. Orange juice is allowed.

If vomiting and/or diarrhea occurs during the course of treatment, nore-dosing of the patient is allowed before the next scheduled dose. Theoccurrence and frequency of any vomiting and/or diarrhea (or increasedstool frequency) within 4 hours after dosing must be noted in the AEssection of the eCRF. In addition, on the days of full PK sampling, theonset time of any episodes of vomiting within the first 4 hourspost-dosing on that day must be noted in the corresponding DoseAdministration Record PK eCRF.

The investigator or responsible site personnel should instruct thepatient to take the study drugs as per protocol (promote compliance).All dosages prescribed and dispensed to the patient and all dose changesand all missed doses during the study must be recorded on the DosageAdministration Record eCRF. Drug accountability must be performed on aregular basis. Patients will be instructed to return unused study drugsto the site at the end of each cycle. The site personnel will ensurethat the appropriate dose of each study drug is administered at eachvisit and will provide the patient with the correct amount of drugs forsubsequent dosing.

For the combination arm of the B-Raf inhibitor of Formula (I) withCompound F only

Instructions for administration on days when a fasting plasma glucosemonitoring is performed: On the days of fasting plasma glucosemonitoring, patients must be fasting overnight for at least 8 hoursprior to the blood collection. A light breakfast/snack may be consumedafter fasting plasma glucose draw. the B-Raf inhibitor of Formula (I)(and Compound F if applicable) may be administered 2 hours afterbreakfast. Patients should continue to fast for 2 hours after theadministration of the B-Raf inhibitor of Formula (I) (and Compound F ifapplicable).

Treatment Duration

Patients may continue treatment with the B-Raf inhibitor of Formula (I)single agent until experiencing unacceptable toxicity, and/or thetreatment is discontinued at the discretion of the investigator orwithdrawal of consent. At disease progression, after the B-Raf inhibitorof Formula (I) single agent treatment, patients will be assigned to acombination treatment according to genetic alterations identified in therelapse biopsy. Patients may continue combination treatment untilexperiencing unacceptable toxicity, disease progression and/or thetreatment is discontinued at the discretion of the investigator orwithdrawal of consent.

Dose Escalation Guidelines

Starting dose rationale.

The B-Raf Inhibitor of Formula (I) Single Agent

The dose for the B-Raf inhibitor of Formula (I), for patients enrolledin the first part of this trial, is set at 450 mg QD, which correspondsto the single agent RP2D. The selection of the starting dose follows theICH S9 guidelines for choosing a starting a dose for a first-in-humantrial conducted in patients with cancer, and is shown in Table 6-2.

The B-Raf Inhibitor of Formula (I) in Combination with Compound B:

The starting dose for the B-Raf inhibitor of Formula (I) plus CompoundB, is set at the B-Raf inhibitor of Formula (I) 600 mg QD and Compound B45 mg BID, or the highest dose combination proven to be safe.

The B-Raf Inhibitor of Formula (I) in Combination with Second Agent(Compound F, Compound H, Compound G or Compound C):

In the second part of this trial, the starting doses for the B-Rafinhibitor of Formula (I) and second agent will be respectively 450 mg QD(RP2D), or the highest last-dose tolerated of the B-Raf inhibitor ofFormula (I) and the highest dose of the second agent allowed by the BLRM(see Table 3).

The RP2D of the B-Raf inhibitor of Formula (I) has been declared at 450mg QD.

Qualitative DDI assessment predicts no significant impact on the B-Rafinhibitor of Formula (I) or Compound F exposure when they areco-administered. Quantitative analysis using SimCYP simulation confirmedthis assessment. Therefore the starting dose for this combo pair isselected to be the currently established RP2D for the B-Raf inhibitor ofFormula (I) and 75% MTD for Compound F: 450 mg QD the B-Raf inhibitor ofFormula (I) and 75 mg Compound F.

Quantitative DDI assessment using Simcyp simulation predicts minimalchanges in the B-Raf inhibitor of Formula (I) exposure whenco-administered with Compound H. At the B-Raf inhibitor of Formula (I)dose of 450 mg, the exposure (Cmax and AUC) of Compound H is expected todecrease by 20-40%. Therefore the starting dose for this combo pair isselected to be the currently established RP2D for the B-Raf inhibitor ofFormula (I) and 60% MTD for Compound H: 450 mg QD the B-Raf inhibitor ofFormula (I) and 75 mg Compound H.

Quantitative DDI assessment using Simcyp simulation predicts 76% and 43%increase in AUC and Cmax of the B-Raf inhibitor of Formula (I),respectively, as well as 54% and 36% decrease in AUC and Cmax ofCompound G, respectively when 450 mg QD the B-Raf inhibitor of Formula(I) and 150 mg BID Compound G are co-administered. In clinic, the B-Rafinhibitor of Formula (I) has been tested at up to 700 mg QD dose and theobserved adverse events are reversible and manageable, therefore thepotential DDI between the two molecules while may result in the B-Rafinhibitor of Formula (I) concentrations higher than the currentlyestablished RP2D, do not pose a risk as the adverse events aremonitorable, manageable and reversible. The starting dose for this combopair is selected to be the currently established RP2D for the B-Rafinhibitor of Formula (I) and 50% MTD for Compound G: 450 mg QD the B-Rafinhibitor of Formula (I) and 150 mg Compound G.

Quantitative DDI assessment using Simcyp simulation predicts 43% and 20%increase in AUC and Cmax of the B-Raf inhibitor of Formula (I),respectively, as well as 43% and 37% decrease in AUC and Cmax ofCompound C, respectively when 450 mg QD the B-Raf inhibitor of Formula(I) and 300 mg Compound C are co-administered. In clinic, the B-Rafinhibitor of Formula (I) has been tested at up to 700 mg QD dose and theobserved adverse events are reversible and manageable, therefore thepotential DDI between the two molecules while may result in the B-Rafinhibitor of Formula (I) concentrations higher than the currentlyestablished RP2D, do not pose a risk as the adverse events aremonitorable, manageable and reversible. The starting dose for this combopair is selected to be the currently established RP2D for the B-Rafinhibitor of Formula (I) and ˜23% of the dose currently tested at the900 mg QD dose level, since the maximum tolerated dose (MTD) has notbeen achieved for Compound C: 450 mg QD the B-Raf inhibitor of Formula(I) and 200 mg Compound C.

In this study, the pharmacokinetics of all combination partners as wellas their active metabolites (if applicable) will be evaluated as soon aspossible at steady-state and compared with those obtained in therespective monotherapy studies for assessment of the potential drug-druginteraction

Before the first patient is dosed with one of the combinations, theBayesian model for this combination will be updated with the most recentdata from the ongoing single agent trial, to confirm that the proposedstarting doses for the B-Raf inhibitor of Formula (I) and second agentare still appropriate (i.e. fulfills the EWOC criteria). If the proposedstarting dose does not meet the criteria, a lower dose combination thatsatisfies the EWOC criteria will be used.

Provisional Dose Levels

Table 3 describes the starting doses and the provisional dose levels ofstudy treatments for the combinations that may be evaluated during thistrial. Additional dose levels not currently specified may be enrolledand additional patients may be enrolled at a dose level already testedif such changes are deemed necessary to provide optimal safety andtolerability, pharmacokinetic, and pharmacodynamic data.

TABLE 3 Provisional dose levels Dose B-Raf Cpd Cpd Cpd Cpd Cpd level*inhibitor B F H G C (mg) QD BID QD QD BID QD  −2** 150 15 30 25  50 100 −1** 300 30 40 50 100 150 1 450 (600 45 60 75 150 200 (starting forCom- (RP2D) dose) pound B arm) 2 450 — 80 100  200 400 3 450 — 100 (MTD)125 (MTD) 250 700 4 450 — — — 300 900 *It is possible for additionaland/or intermediate dose levels to be added during the course of thestudy Cohorts may be added at any dose level below the MTD in order tobetter understand safety, PK or PD. **Dose level −1 and −2 will also beused for patients requiring a dose reduction from the starting doselevel. No dose reduction below dose level −2 is permitted for thisstudy.Implementation of Dose Escalation Decisions

The decision to escalate the dose of the second agent will occur afterevaluation of the individual patient tolerability of the dualcombination during the first 21 days of the cycle.

To implement dose escalation decisions, the available toxicityinformation (including adverse events and laboratory abnormalities thatare not DLTs), the recommendations from the BLRM, and the available PKand PD information will all be evaluated during a dose decision. Drugadministration at the next higher dose level may not proceed until theinvestigator receives written confirmation indicating that the resultsof the previous dose level were evaluated and that it is permissible toproceed to a higher dose level. If a decision is made to escalate to ahigher dose level but one or more additional patient(s) treated at thepreceding dose level experiences a DLT during the first cycle oftreatment, then the BRLM will be updated with this new informationbefore any additional patients are enrolled at that higher dose level.

The dose escalation process will be implemented stepwise and willproceed with cohorts of 3 patients. Only the second agent, Compound F,Compound H, Compound G, or Compound C will be escalated according to theBLRM.

Intra-patient dose escalation is not permitted at any time within thepart I of treatment with the B-Raf inhibitor of Formula (I) singleagent.

Intra-patient dose escalation for second agent is permitted during thesecond part with the combination treatment with the exception ofpatients in the the B-Raf inhibitor of Formula (I)+Compound B arm, whowill be treated at the declared RP2D of the combination of the B-Rafinhibitor of Formula (I) and Compound B (45 mg BID)

In order for a patient to be treated at a higher dose of Compound F,Compound H, Compound G, or Compound C, he or she must have tolerated thelower dose combination for at least 1 cycle of therapy (e.g., he or shemust not have experienced at the lower dose pair originally assigned atoxicity of CTCAE grade ≧2 for which relationship to study drugs cannotbe ruled out). Moreover, the new, higher dose pair with which thepatient is to be treated must meet the modified EWOC criteria used forintra-patient escalation (add ref to section).

Newly enrolled patients in the next cohort will start treatment at thedose decided at the last dose escalation conference. The Bayesianlogistic regression model (BLRM) and the intra-patient dose boundarieswill then be updated for the next cohort.

Treatment Interruption and Treatment Discontinuation

If a patient requires a dose delay of >21 consecutive days of the B-Rafinhibitor of Formula (I), Compound B, Compound F, Compound H, CompoundC, or Compound G from the intended day of the next scheduled dose, thenthe patient should be discontinued from the study treatment. Inexceptional situations, if the patient is clearly benefiting from thestudy treatment (i.e. stable disease, partial response, completeresponse), and in the opinion of the investigator no safety concerns arepresent, the patient may remain on the study treatment at a dose leveladjusted based on safety.

Molecular Pre-Screening

Molecular Pre-Screening Informed Consent

The molecular pre-screening informed consent must be signed prior to anystudy-related molecular pre-screening procedure (not applicable if themutational status of BRAF was already assessed outside of the study).This applies to Part 1 patients only.

BRAF Mutational Status on Fresh or Archival Biopsy

To enter the screening phase of the study, patients must have writtendocumentation of BRAF V600 mutation, which should be obtained locally ona fresh tumor biopsy (preferred) or the most recent archival tumorsample available. The molecular pre-screening informed consent must besigned prior to any study-related molecular pre-screening procedure (notapplicable if the mutational status was already assessed outside of thestudy).

Once the mutation of the BRAF V600 codon (e.g. V600E/K/D/R) is confirmedby the designated local laboratory and documented by the site, thepatient may begin the screening procedures.

Treatment Period

Treatment period is divided into two parts:

Part I: BRAF inhibitor naïve patients will be dosed continuously withthe B-Raf inhibitor of Formula (I) on 21-day (3 calendar weeks) cyclesbeginning on Day 1 of Cycle 1. There will be no scheduled break betweencycles. Patients will receive the B-Raf inhibitor of Formula (I) singleagent until initiation of combination treatment after progression ofdisease or unacceptable toxicity occurs, whichever comes first.

Part II: Patients who received the B-Raf inhibitor of Formula (I) for atleast one 21-day cycle and who progressed will enter in the Part II toreceive a treatment combination of the B-Raf inhibitor of Formula(I)+second agent, based upon the genetic alterations identified in thetumor biopsy at relapse.

There is no fixed treatment duration; patients may continue treatmentwith the B-Raf inhibitor of Formula (I) single agent until combinationtreatment and during first disease progression, unacceptable toxicityoccurs that precludes any further treatment and/or treatment isdiscontinued at the discretion of the investigator or by patient refusal(withdrawal of consent). At the time of first disease progression, oncebiopsy's analysis results are known, patients may initiate combinationtreatment with the B-Raf inhibitor of Formula (I)+second inhibitor untilsecondary disease progression, unacceptable toxicity occurs thatprecludes any further treatment and/or treatment is discontinued at thediscretion of the investigator or by patient refusal (withdrawal ofconsent) If a patient remains on study although the patient required adose interruption of >21 days, because the patient had experiencedobjective evidence of clinical benefit and in the opinion of theinvestigator it is in the best interest of the patient to remain onstudy.

Bayesian Logistic Regression Model

An adaptive BLRM guided by the EWOC principle will guide the doseescalation of each of the study drugs (Compound F, Compound H, CompoundG or Compound C) combined with the B-Raf inhibitor of Formula (I) to itsrespective MTD(s)/RP2D(s). For each combination, a 5-parameter BLRM forcombination treatment will be fitted on the Cycle 1 dose-limitingtoxicity data (i.e. absence or presence of DLT) accumulated throughoutthe dose escalation to model the dose-toxicity relationship of CompoundF, Compound H, Compound G or Compound C given in combination with theB-Raf inhibitor of Formula (I).

The definition of the BLRM's, the prior distributions for the modelparameters (based on currently available information about the targetedagents) and the associated prior distribution of DLT rates are providedin Appendix 1.

Dose Recommendation

Dose recommendation for the combination partner is conditional to thedose of the B-Raf inhibitor of Formula (I) which may differ betweenpatients entering Part II. This recommendation will be based onposterior summaries of the DLT rate including the mean, median, standarddeviation, 95%-credibility interval, and the probability that the trueDLT rate for each dose combination lies in one of the followingcategories:

[0%, 16%] under-dosing

[16%, 35%] targeted toxicity

[35%, 100%] excessive toxicity

Following the principle of EWOC, after each cohort of patients therecommended dose combination is the one with the highest posteriorprobability of DLT in the target interval [16%, 35%] among the dosesfulfilling the overdose criterion that there is less than 25% chance ofexcessive toxicity. In addition, the maximum inter-cohort combined doseescalation across the two study drugs is limited to 100%, where 100%refers to the sum of the relative escalation for each of the studydrugs, i.e. 0% and 100% for the B-Raf inhibitor of Formula (I) (whichdose cannot exceed 450 mg) and for the second targeted agent (whichcannot be escalated beyond its s.a. MTD/RP2D if available),respectively.

The Intra-patient dose escalation of the combination partner will belimited to 50% and will be guided by the BLRM according to the followingmodified EWOC criterion which reflects individual patient tolerability:a patient will be able to intra-escalate to a dose for which there isless than a 40% chance of excessive toxicity. Furthermore, iftreatment-related toxicities of CTCAE grade 2 are observed in 2 or morepatients at a dose level or if any patient experiences a grade 3 orgreater toxicity, then the increase in dose of the combination partnerwill be ≦25% for any subsequent increase in dose.

A clinical synthesis of the available toxicity information (includingAEs that are not DLTs), PK, PD, and efficacy information as well as therecommendations from the Bayesian model will be used to determine thedose combination for the next cohort at a dose-escalation conference.The Investigators and trial personnel will be involved in the decisionmaking.

The model for any combination will be re-evaluated before enrollment ofany additional patients to the cohort if the first 2 evaluable patientsin the cohort experience DLT before the enrollment of the 3rd patient.The final recommended MTD/RP2D for each combination will be based onconsiderations of the recommendation from the BLRM, and on an overallassessment of safety taking into consideration tolerability data fromsubsequent cycles at all different dose combinations tested.

EXAMPLE 2 Materials and Methods

Compound stocks are prepared in DMSO at a final concentration of 10 mM.Working stocks are serially diluted in the appropriate cell culturemedium in 3-fold increments to achieve final assay concentrationsranging from 2.71M to 1.2 nM.

Cell Lines, Cell Culture, Cell Viability Measurements

A-375 and WM-266-4 cells were purchased from American Type CultureCollection (ATCC). The A-375 cells were cultured in DMEM medium (ATCC)and the WM-266-4 cells were cultured in EMEM medium (ATCC) bothsupplemented with 10% fetal bovine serum (Gibco) and incubated at 37°C./5% CO2. The cell lines engineered to express commonly occurringalleles indicative of resistance were acquired from Novartis-Emeryville.These resistant models include, A-375 cells expressing mutant MEK1P124L,truncated p61-BRAFV600E, or mutant NRASQ61K, and WM-266-4 cellsexpressing mutant MEK1C121S, truncated p61-BRAFV600E, or mutantNRASQ61K. These cells were cultured in the appropriate parental mediumwith selection marker G418 and in the presence of 5 uM LFE158 (MEKmutants) or LIH720 (truncated p61-BRAFV600E).

Plate Layout, Cell Dispensing and Compound Addition

For screening, cells were seeded in 80 ul of medium in 384-well plates(Thermo Scientific, cat#4332) at 500 (A-375) or 750 (WM-266-4) celldensities per well using a MultiDrop Combi (Thermo-Fisher) with an8-channel standard cassette. To promote an even distribution of cellsacross the entire well, cells were briefly centrifuged at 1000 RPM andincubated at room temperature 30 minutes. All plates were incubated at37° C., 5% C02 for 24 h prior to compound addition. Compound stock wasfreshly prepared in the appropriate culture medium, and added using aPAA robot equipped with a 200 nl pin tool. In a minimum of threereplicate wells, single agent and combination effects after 72 hours,were assessed by both quantification of cellular ATP levels via CellTiter Glo (Promega) according to the manufacturer's protocol and bymicroscopy imaging. For imaging, cells were fixed to the plates andpermeabilized with a solution of 10% PFA, 0.3% TX-100 in PBS via aWellMate dispenser with controlled dispensing speeds. Cell nuclei werestained with Hoechst 33342 (H3570, Invitrogen), and all necessarywashing steps were performed by a BioTek washer.

Automated Image Analysis

Images from the InCell Analyzer 2000 (GE Healthcare, 28-9534-63) were inTIFF format and had a size of 2048×2048 pixels, capturing the whole wellof a 384-well plate. An automated image analysis pipeline wasestablished using custom-made scripts in the open-source, statisticalprogramming language R, and functions of the BioConductor packageEBImage. The goal was to quantify the number of viable nuclei (cells)per well as an approximation for cell viability. The pipeline wascomprised of seven steps: (I.) smoothing of the image to reduce thenumber of intensity peaks, (II.) application of a thresholding functionto separate the foreground (signal) from the background (noise), (Ill.)identification of local maxima in the foreground that serve as seeds forthe nuclei, (IV.) filtering of local maxima in close proximity, (V.)propagation of the nuclei from remaining local maxima, (VI.) andextraction of object features from the propagated nuclei (numbers ofnuclei, size features and intensity features). As a last step (VII.), toexclude debris (e.g. fragmented nuclei) from counting, objectsidentified in DMSO- and Staurosporin-treated wells were used to obtainfeature distributions for viable and fragmented nuclei, respectively.These were used to set cut-offs differentiating between viable andfragmented nuclei. The number of fragmented nuclei was subtracted fromthe total number of identified objects and the result was reported asfinal count for that well.

Data Normalization

Data comprised triplicate measurements for each treatment (compound)condition, 42 replicates of DMSO-treated wells, and duplicates ofStaurosporin-treated wells. The data was normalized to the median of theDMSO measurements and summarized by calculating the median of thetriplicates. Data was imported into Chalice to calculate compoundsynergies.

TABLE 4 Chart of Single Agent IC50 Values and Combination Synergy Scoresas determined using ATP-based CTG assay Cpd. of Combination Parent CellFormula (I) Cpd. B Lowe Excess Line Resistant Allele IC50 (nM) IC50 (nM)Synergy A-375 — 4 51 3.0 A-375 MEK1^(P124L) 333 >2700 7.8 A-375 p61BRaf^(V600E) 576 961 4.6 A-375 NRAS^(Q61K) 134 206 4.3 WM-266-4 — 2 504.2 WM-266-4 MEK1^(C121S) 35 821 5.4 WM-266-4 p61 BRaf^(V600E) 906 >27005.8 WM-266-4 NRAS^(Q61K) 1122 >2700 5.1

TABLE 5 Chart of Single Agent IC50 Values and Combination Synergy Scoresas determined using microscopy assay Cpd. of Combination Parent CellFormula (I) Cpd. B Lowe Excess Line Resistant Allele IC50 (nM) IC50 (nM)Synergy A-375 — 4 57 2.4 A-375 MEK1^(P124L) 300 >2700 9.3 A-375 p61BRaf^(V600E) 849 969 5.9 A-375 NRAS^(Q61K) 133 150 4.6 WM-266-4 — 3 774.7 WM-266-4 MEK1^(C121S) 58 1210 6.3 WM-266-4 p61 BRaf^(V600E)933 >2700 6.8 WM-266-4 NRAS^(Q61K) 868 >2700 4.5

EXAMPLE 3

Single agent and combinatorial effects on proliferation of inhibitors ofRAF (Compound of Formula (I)) and PIK3Cα (Compound X) kinases in sevenBRAF-mutant CRC-derived cell lines. All cell lines express the BRAFV600Eprotein. Cells harboring known or putative activating mutations in thePI3Kα gene are marked with a (*) and cells with PTEN loss marked with a(#). Cell proliferation was measured in 72 hr cell titer Glo™ assays andall results supplied are the result of at least triplicate measurements.Shown are single agent IC50 values for the Compound of Formula (I) andCompound X. Synergy score (SS) measurements as well as the combinationindex (CI50) at the 50% effect level are given for each combination) inTable 6. Interactions were deemed synergistic when SS values were ≧2.0and CI values were ≦0.5. Interactions were deemed additive/synergisticwhen either SS values were ≧2.0 but CI values were >0.5 or SS valueswere <2.0 but CI values were ≦0.5. Interactions where termed additivewhen SS values were <2.0 and CI values were >0.5. Synergy calls aregiven in the “effect description” columns.

TABLE 6 Combinations of Cpd. Form (I) with Cpd X in BRAF^(V600E) mutantCRC cell lines Cell Line Cancer Form (I) Cpd. X Combination Name TypeIC₅₀ [nM] IC₅₀ [nM] SS CI₅₀ Effect Description SW1417 CRC 235 >2700 2.46± 0.06 0.27 ± 0.04 Synergy COLO 205 CRC 5.0 >2700 3.80 ± 0.06 0.69 ±0.01 Additive/Synergy LS411N CRC 18 >2700 2.76 ± 0.07 0.49 ± 0.03Synergy CL-34 CRC 30 >2700 4.48 ± 0.1  0.57 ± 0.03 Additive/SynergyHT-29* CRC 49 >2700 4.31 ± 0.06 0.21 ± 0.02 Synergy RKO* CRC 1965 >27005.24 ± 0.05 0.22 ± 0.01 Synergy SNU-C5* CRC >2700 >2700 2.44 ± 0.1  0.47± 0.07 Synergy OUMS-23^(#) CRC >2700 >2700 0.64 ± 0.06 N/C Additive

EXAMPLE 4

This Example studies the effect of the Compound of Formula (I) andCompound F as single agents and in combination on the growth of theHT-29 and RKO cell line models in vivo. Concentration and dosingschedules for the inhibitors were 50 mg/kg q.d (Compound of Formula(I)), and 32.7 mg/kg q.d (Compound F). All compounds were dosed incombination as they were as single agents. Dosing was stopped at 28 daysin the HT-29 model and after 21 days in the RKO model. The results arereported in FIGS. 1 and 2, respectively.

EXAMPLE 5

All cell lines were purchased from ATTC (SK-MEL-5, SK-MEL-24, UACC-62,COLO 741, COLO-800, WM-266-4, Colo205, LS411N, SW 1417), ECACC (MDST8),DSMZ (CL-34) and NCI (LOX IMVI). Cells were cultured either in RPMI1640(ATCC, Catalog number 30-2001) or DMEM (ATCC, Catalog number 30-2002)supplemented with 10% (or 20% for CL-34 cells) FBS (GIBCO, Catalognumber 10099-141) according to vendor recommendations. Cell lines werecultured in 37° C. and 5% CO2 incubator and expanded in T-75 flasks. Inall cases cells were thawed from frozen stocks, expanded through ≧1passage using 1:3 dilutions, counted and assessed for viability using aViCell counter (Beckman-Coulter), prior to plating in 96-well or 6-wellplates. To split and expand cell lines, cells were dislodged from flasksusing 0.25% Trypsin-EDTA (GIBCO, Catalog number 25200). All cell lineswere determined to be free of mycoplasma contamination as determined bya PCR detection methodology performed at Idexx Radii (Columbia, Mo.,USA) and correctly identified by detection of a panel of SNPs.

The Compound of Formula (I) and Compound H dissolved in 100% DMSO(Cellgro, catalog number 25-290-CQC) at concentrations of 10 mM andstored at −200° C. until use. Compounds were arrayed in 2 ml deep96-well plates (Greiner bio-one, catalog number 780271) serially diluted3-fold seven times yielding concentration ranges from 22 nM to 16200 nM.Recombinant Human FGF basic was purchased from R&D system (Catalognumber 233-FB), and reconstituted at 50 μg/ml in sterile PBS. It wasused at a fixed concentration 100 ng/ml in all experiments.

For CellTiter-Glo™ assays, cells were dispensed into tissue culturetreated 96-well plates (Costar, catalog number 3904) with a final volumeof 80 μL of medium and at density of 3000 cells per well. 12 to 24 hrsafter plating, 20 μL of each compound dilution series were transferredto plates containing the cells, resulting in compound concentrationranges of 2700 nM to 3.7 nM by 3-fold dilutions and a final DMSOconcentration of 0.16%. The total volume in each well was 120 μL. Plateswere incubated for 72 hrs and the effects of compounds on cellproliferation was determined using the CellTiter-Glo™ Luminescent CellViability Assay (CTG, Promega) and a Victor™ X4 plate reader (PerkinElmer). For real-time growth assays cells were seeded into xCELLigenceE-plates (Roche catalogue number 05232368001) at a density of 4000 cellsper well in a total of 90 μl of media and 24 hrs after plating, 11 μl ofmedia with or without compound was added to the wells. 2-4 replicatewells were plated for all cell lines and treatment groups with theexception of LGX818+FGF2 treated COLO 741 cells (N=1). Where indicatedfinal concentrations of compounds and growth factor were 1 uM forCompound H, 100 ng/ml for FGF2, and either 100 nM (SK-MEL-5) or 500 nM(COLO 741) for the Compound of Formula (I). Cells were continuouslymonitored every two hours for seven days with the xCELLigence real-timeimpedance based cell analyzer. Impedance was measured using theelectrodes on the E-plates, with increasing surface area coverage ofcells creating greater electrode impedance. Electrode impedance wasdisplayed as cell index values, and used as a proxy for cell viabilityand number. In all cases cell index values were normalized to atimepoint immediately following the addition of compound.

For Western analysis, cells were plated in 6-well (Costar, catalognumber 3506) plates at a density of 5×105 cells per well in 2.0 ml ofcomplete culture medium. Twelve to 24 hrs after plating, cells weretreated with the various compounds, and in all experiments the Compoundof Formula (I), Compound H, and FGF2 were used at final concentrationsof 100 nM, 1.0 uM, and 100 ng/ml, respectively. Cells were harvested 2,and 24 hrs following the addition of compound in freshly prepared celllysis buffer (Cell Signaling Technology Catalog number 9803);supplemented with both phosphatase (PhosStop, Roche Catalog number04906845001) and protease inhibitors (Roche, catalog number11697498001). Proteins from cell lysates were separated byelectrophoresis though a NuPAGE 4-12% Bis-Tis midi gel (Novex, catalognumber WG1402BX10), transferred to nitrocellulose membranes, which weresubsequently incubated with antibodies from Cell Signaling Technology(Danvers, Mass., USA) recognizing p-AKT (S473, Catalog number 4058),total AKT (Catalog number 2920), p-ERK1/2 (T202/Y204, catalog number9101), total ERK1/2 (catalog number 9107), p-MEK1/2 (S217/221, catalognumber 9121) and β-actin (Ambion, catalog number AM4302). Western blotswere visualized following incubation with IRDye 680RD Goat anti-RabbitIgG (Li-Cor, Catalog number 926-68071) and IRDye 800 CW Goat anti-MouseIgG (Li-Cor, Catalog number 926-32210) and scanning with an OdysseyInfrared Imager System (Li-Cor, Lincoln, Nebr., USA).

To determine whether FGFRs can rescue a subset of BRAFV600E mutantmelanoma cell lines treated with the Compound of Formula (I), itsanti-proliferative effects were examined in eleven T1799 mutant BRAFcell lines either in the presence or absence of the FGFR-activatingligand FGF2. In the absence of FGF2, IC50 values in six melanoma, andfive CRC-derived cell lines ranged from 3.0 to 470 nM and 4.0 to 185 nM,respectively (Table 7). IC50 values measured for six of the cell lineswere unaffected by the presence of FGF2, however, for the remaining fivecell lines response to the Compound of Formula (I) was either greatlydiminished (e.g. CL-34) or completely abolished (e.g. Is 411N). Thus,the presence of FGF2 is able to rescue a set of BRAFV600Emelanoma-derived cell lines from the anti-proliferative effects of theB-Raf inhibitor of Formula (I). Furthermore, similar effects are alsoobserved in BRAFV600E mutant cell lines derived from CRC tumors.

TABLE 7 LGX818 + Cell Line Cancer LGX818 FGF2 Name type BRAF PTENIC₅₀[nM] IC₅₀[nM] SK-MEL-5 skin mut wt 15 >1000 SK-MEL-24 skin mut mut470 369 UACC-62 skin mut mut 2.0 4 COLO 741 skin mut wt 53 2652 COLO-800skin mut wt 9 12 WM-266-4 skin mut** mut 3 4 CL-34 CRC mut wt 38 730.0Colo 205 CRC mut wt 4 5 LS411N CRC mut wt 185 9232 MDST8 CRC mut*unknown 141 10000 SW1417 CRC mut wt 165 364

Single agent IC50 values for the Compound of Formula (I) with or without100 ng/ml FGF2 in a panel of BRAF T1799 mutant melanoma and CRC celllines. Mutant (mut) and wildtype (wt) status was determined frompublished data. All BRAF mutations resulted in the V600E substitution,except in the cases of MDST8 (mut*) and WM-266-4 (mut**) which haveV600K and V600D respectively. PTEN mut designations represent a summarycall based on mutation, gene copy number, and mRNA expressioninformation for the PTEN gene.

To determine whether rescue of BRAFV600E melanoma cell lines by FGF2 wasdependent on FGFR signaling, we examined whether the selective FGFRinhibitor Compound H could prevent FGF2-mediated rescue. Two cell lineswhich were rescued to varying degrees by FGF2 (COLO 741, and SK-MEL-5,Table 8-1) were cultured in media containing the Compound of Formula (I)and FGF2 either in the presence or absence of Compound H and growthmeasured in real time.

Consistent with the earlier IC50 data, the Compound of Formula (I)suppressed the growth of both cell lines, and this growth suppressionwas abrogated by the addition of FGF2 (FIG. 3). As a single agentCompound H did not affect the proliferation of either of the cell lines(panels A, not shown for SK-MEL-5), and when combined with the Compoundof Formula (I) in the absence of FGF2 did not contribute to its singleagent activity. When combined with the Compound of Formula (I) in thepresence of FGF2, Compound H restored anti-proliferative effects tolevels observed in the absence of FGF2. These results indicate that FGF2mediated rescue can be prevented via the addition of the selective FGFRinhibitor Compound H.

To investigate whether either restored MAPK or activated PIK3C signalingmight underlie FGF2-mediated rescue the effects of FGF2 on MAPK andPIK3C signaling were examined via western-blot analysis ofphosphorylated MEK1/2 (MAP2K1/2), ERK1/2 (MAPK1/2) and AKT1/2/3. Asdemonstrated in FIG. 4, incubation of COLO 741 and SK-MEL-5 cells withthe Compound of Formula (I) resulted in marked suppression of MAPKsignaling at both 2 and 24 hours following compound addition as judgedby reductions in levels of both phosphorylated MEK and ERK.Phosphorylated levels of AKT were unaffected at 2 hours, but showedmodest decreases at 24 hrs in COLO 741 cells. In contrast, neither FGF2nor Compound H when added as single agents affected signaling at either2 or 24 hours. When FGF2 and the Compound of Formula (I) were combined,minimal changes in signaling relative to the Compound of Formula (I)alone were observed two hours after treatment (although slight increasesin p-ERK were observed in SK-MEL-5 cells), however, levels of bothphosphorylated MEK and ERK had been largely, although not completely,restored by 24 hrs. Lastly, the addition Compound H to the Compound ofFormula (I)/FGF2 combination completely abolished the FGF2-inducedchanges in MAPK and PIK3C signaling. These data strongly suggest thesuppression of the B-Raf inhibitor's anti-proliferative effects by FGF2results from a re-activation of MAPK signaling, and indicate thatCompound H is able to completely suppress these FGF2 induced signalingchanges.

EXAMPLE 6

Objective: To evaluate the efficacy of the combination of the Compoundof Formula (I) and the CDK 4 inhibitor Compound C in the HMEX1906primary melanoma model that is grown in the presence of and is resistantto 5 mg/kg the Compound of Formula (I) (HMEX1906-R5)

Drug formulation: Compound C is formulated in 0.5% MC/0.5% Tween80 andthe Compound of Formula (I) is formulated in 20% PEG300/3% ETPGS.

Tumors are chopped/minced into cell line like suspension (tumorshomogenized). 7 mL of matrigel added and 1.5 mL of HBSS. Suspensionwarmed in palm until Matrigel is thick and implanted with a 18G needles.c right flank of female nude mice.

The mice were assigned to the following groups at 18 days post implantwith an average tumor volume of 266 mm³ and average body weight of 25grams.

Groups: 10 mice/group, route PO, dose volume 0.2 mL

Group 1: Vehicle, 0 mg/kg bid×14

Group 2: Compound C, 250 mg/kg qd×21

Group 3: Compound of Formula (I), 5 mg/kg bid×21

Group 4: Compound C 250 mg/kg qd×21+Compound of Formula (I) 5 mg/kgbid×21

Results:

Mean change Mean of tumor change of Mean volume vs tumor change controlRegres- volume of body (T/C) sion (mm3 ± weight Survival Group (%) (%)SEM) (% ± SEM) (Survivors/Total) 1 100 — 2092 ± 154  4.2 ± 2.6  7/10* 24 — 86 ± 26 5.3 ± 1.4 10/10 3 39 — 807 ± 106 3.5 ± 1.1 10/10 4 — 64.32−170 ± 45  7.1 ± 1.6 10/10 *3 mice were euthanized due to large tumor

The invention claimed is:
 1. A therapeutic combination consistingessentially of a B-Raf inhibitor of Formula (I)

or a pharmaceutically acceptable salt thereof; and a second inhibitorwhich is a PI3 kinase inhibitor of5-(2,6-di-morpholin-4-yl-pyrimidin-4-yl)-4-trifluoromethyl-pyridin-2-ylamine(Compound F) or (S)-pyrrolidine-1,2-dicarboxylic acid 2-amide1-({4-methyl-5-[2-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl}-amide)(Compound X), or a pharmaceutically acceptable salt thereof, forseparate, simultaneous or sequential administration.
 2. The therapeuticcombination of claim 1, wherein the PI3 kinase inhibitor is5-(2,6-di-morpholin-4-yl-pyrimidin-4-yl)-4-trifluoromethyl-pyridin-2-ylamine(Compound F), or a pharmaceutically acceptable salt thereof.
 3. Thetherapeutic combination of claim 1, wherein the PI3 kinase inhibitor is(S)-pyrrolidine-1,2-dicarboxylic acid 2-amide1-({4-methyl-5-[2-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl}-amide)(Compound X), or a pharmaceutically acceptable salt thereof.